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Modification of 4q35 and muscular gene expression in fetuses carrying a shortened D4Z4 array linked to FSHD
Natacha Broucqsault, Stéphane Roche, Julia Morere, Marie-Cécile Gaillard, Nicolas Levy, Karine Nguyen, Frédérique Magdinier
To cite this version:
Natacha Broucqsault, Stéphane Roche, Julia Morere, Marie-Cécile Gaillard, Nicolas Levy, et al..
Modification of 4q35 and muscular gene expression in fetuses carrying a shortened D4Z4 array linked
to FSHD. 18th International Congress of The World Muscle Society, Oct 2013, Pacific Grove, CA,
United States. pp.824, �10.1016/j.nmd.2013.06.643�. �hal-01907599�
1Centre de Re´fe´rence Neuromusculaire Nantes Angers, CHU Nantes, Nantes, France; 2Centre de Re´fe´rence Neuromusculaire Nantes Angers, Atlantic Gene Therapy – Biotherapy Institute for Rare Diseases, Nantes, France; 3Faculte´ de Me´decine Nantes, Nantes, France
FSHD is one of the most frequent forms of muscular dystrophy char- acterized by an early involvement of facial and scapular muscles with even- tual spreading to pelvic and lower limb muscles. Few studies have been dedicated to sleep disorders in FHSD. Our objective was to explore subjec- tive sleep quality in FSHD patients. Adults with genetically proven FSHD were included in the study. They were compared to a control group of sub- jects with no myopathy, no past neurological disease nor instable chronic disease history. The Pittsburgh sleep quality index (PSQI), the Epworth sleepiness scale (ESS), the clinical severity scale (CSS), the fatigue severity scale and the hospital anxiety and depression scale were used to evaluate the severity of the sleep disorder, the subjective sleep quality, the prevalence of daytime sleepiness, the anxiety and the depression in patients and con- trols. Pulmonary function and MRC score were also assessed. Mann With- ney test, Spearman correlation and Fisher test were used for the statistics analysis. 22 FSHD and 36 controls were included in the study. Both groups were similar as for age, sexe, body mass index and respiratory support. 50%
of FSHD patients presented with vital capacity <80%. The FSHD popula- tion had a significantly worse sleep quality (PSQI) than the control group (p< 0.01), with loss of subjective sleep quality, nocturne pain, bad dreams and day dysfunction due to sleepiness. Altered sleep quality was not corre- lated with the severity of the disease or with respiratory deficiency but was related with anxiety (p= 0.04). The latter was also correlated with depres- sion, fatigue and bad dreams. Surprisingly, we noticed a negative correla- tion between ESS and CSS: the loss of activity in severe patients could result in harmony with sleep needs.
FSHD patients have altered subjective sleep quality as compared with control population. Anxiety mainly accounts for this, while severity of the disease and respiratory involvement do not affect it.
http://dx.doi:10.1016/j.nmd.2013.06.642
P.16.6
Modification of 4q35 and muscular gene expression in fetuses carrying a shortened D4Z4 array linked to FSHD
N. Broucqsault, S. Roche, J. Morere, M.C. Gaillard, N. Levy, K. Nguyen, F. Magdinier
Inserm, UMR_S910, Marseille, France
Facio-Scapulo-Humeral Dystrophy (FSHD) is an enigmatic pathol- ogy. This autosomal dominant disorder is linked to deletion within a D4Z4 macrosatellite in the subtelomeric 4q35 region. The gene product leading to the disease has not been clearly identified and epigenetic changes are likely key players in the disease since beside reduction in the number of repeats, D4Z4 is hypomethylated in FSHD. Within D4Z4, DUX4 has been found upregulated in patients. Different DUX4 tran- scripts have been described Production of a long transcript encompassing the DUX4 sequence and a region distal to D4Z4 encoding a toxic protein has been proposed as the cause of disease. A causal link between DUX4 expression and D4Z4 hypomethylation subsequent to array shortening has been proposed but never firmly established. We analyzed DUX4 expression as well as the expression of 4q35 genes and muscular markers in FSHD and non-FSHD biopsies during fetal development and in adults in a large cohort of samples. We highlight several genes whose expression differs between control and FSHD samples. Furthermore, we detected DUX4 transcripts in both groups, in muscle but also in other tissues indi- cating that expression of the long transcript is not restricted to FSHD muscles. Using FSHD and control myoblasts, we showed that DUX4 expression is induced by hypomethylation after knock-down of DNA methyltransferases, independently of D4Z4 array shortening. Our result tends toward a stochastic activation DUX4 transcription rather than a
muscle-specific expression pattern in FSHD patients. The mechanism pre- cluding onset and progression of FSHD remains highly controversial and still debated. Our work is the first to uncover changes in gene expression in fetuses carrying a D4Z4-linked 4q35 defect. These results are important for understanding FSHD but also in general, to understand how epige- netic mechanism modulate the transcription of repetitive DNA sequences in the human genome especially in the human diseases.
http://dx.doi:10.1016/j.nmd.2013.06.643
P.16.7
Length dependent telomere looping affects long-distant gene expression (5 Mb) in FSHD
J.D. Robin1, A.T. Ludlow1, G. Stadler1, F. Magdinier2, W.E. Wright1, J.W. Shay1
1UT Southwestern Medical Center, Cell Biology, United States; 2Faculte de Medecine de La Timone, Marseille, France
The genetic locus of Facioscapulohumeral Muscular Dystrophy (FSHD) is only25–50 kb from the telomere of Chr.4q, and shows an age-related onset of symptoms. This suggested that Telomere Position Effects (TPE) might regulate its poorly understood pathogenetic mecha- nism. FSHD patients show a reduction in the number of 3.3 kb D4Z4 repeats on at least one copy of chromosome 4. We first showed that contin- uous spreading of telomeric heterochromatin (classic TPE) could extend at least 100 kb into the subtelomeric region and increased Dux4-fl expression
>10-fold when telomeres were short. We then examined whether telomere looping existed/influenced gene expression over much larger distances. We adapted the Hi–C technique to study the 4q region in an unbiased fashion.
We established clonal myoblast cell lines containing a floxable hTERT that we removed at different times to initiate telomere shortening, producing isogenic subclones with different telomere lengths. We identified multiple novel long-range interactions at 4q associated with telomere shortening.
We validated one interaction using 3C and 3D co-FISH experiments. A 4.8 Mb DNA loop present in FSHD myoblasts with long telomeres (20 Kb) and in cells obtained from unaffected age matched siblings (regard- less of telomere length) was lost on the FSHD contracted allele when telo- meres became shorter (9 Kb,long before telomeres became short enough to induce replicative senescence). Intriguingly, interactions involved several putative FSHD genes and a gene supposedly expressed only in cardiac mus- cle. Droplet-digital PCR (ddPCR) demonstrated changes in the abundance of different exon-pairs for a variety of candidate genes affecting FSHD in cells with short vs long telomeres, indicating telomere looping was at least altering splicing patterns if not overall transcription. These results suggest a role for telomere looping in FSHD pathology that could explain much of the age-related onset and variability in this disease.
http://dx.doi:10.1016/j.nmd.2013.06.644
P.16.8
Does endurance training and protein supplementation improve fitness in patients with Facioscapulohumeral Muscle Dystrophy (FSHD)?
G. Andersen, K.P. Prahm, J. Dahlquist, G. Citirak, J. Vissing
Rigshospitalet, University of Copenhagen, Neuromuscular Research Unit, Department of Neurology, Copenhagen, Denmark
In healthy subjects, regular exercise is beneficial for mental and phys- ical health, and post-exercise protein supplementation has been shown to enhance the physiological responses to training. FSHD is a muscular dys- trophy characterized by slow progression and asymmetry of muscle weak- ness, often starting in facial, shoulder, humeral and calf muscles.
Moderate, and even high-intensity exercise, has been shown to be well tol- erated in these patients. In this double-blinded, placebo-controlled study,
824 Abstracts / Neuromuscular Disorders 23 (2013) 738–852
